Megakaryocyte differentiation factor

ABSTRACT

A novel megakaryocyte differentiation factor, for example, consisting essentially of SEQ ID NO: 30; DNA coding for the megakaryocyte differentiation factor, an expression vector comprising the DNA, a host transformed with the expression vector, and a process for production of the megakaryocyte differentiation factor using the host. 
     The megakaryocyte differentiation factor accelerates differentiation of megakaryocytes in the presence of IL-3, and acts as a thrombopoietin, and therefore an effective medicament to various diseases involving a decrease in platelete.

This application is a continuation, of application Ser. No. 08/091,028filed Jul. 14. 1993 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a megakaryocyte differentiation factor,gene coding for the factor and a process for production thereof. Themegakaryocyte differentiation factor is useful as a hemopoieticstimulating factor for megakaryocyte-platelet lineage.

2. Description of Related Art

It is well known that various hemopoietic factors inducing the growthand differentiation of blood cells are involved in a process fromhemopoietic stem cells to mature blood cells.

Although the life time of platelet is as short as 9 to 10 days,concentration of platelets in the blood is maintained rather constantduring the stationary state. Moreover, when the number of platelets isartificially reduced by one of various available method in anexperimental animals, the number of the platelets recovers in the bloodin a few days. From these facts, it is supposed that factors whichstimulate formation of platelets are present, and so far a great efforthas been made to identifycation of the factors.

It is considered that at least two regulatory factors are involved inthe megakaryocyte-platelet hemopoietic lineage. The first factor byitself stimulates formation of megakaryocyte colonies and is called amegakaryocyte colony stimulating factor. The second factor by itselfdoes not have an activity to stimulate formation of megakaryocytecolonies, but in combination with the first factor, increases the numberof megakaryocyte colonies and stimulates the differentiation thereof,and is called a megakaryocyte potentiator.

The former includes interleukin 3, and granulocyte/macrophage colonystimulating factor, and the latter includes erythropoietin, macrophagecolony stimulating factor, interleukin 6, 7 and 11, LIF, and the like.Some of these factors actually exhibit in vivo effect of increasing thenumber of platelets or shortening the time required to recover thenumber of platelets (Hideaki Mizoguchi: Tanpakushitsu Kakusan Koso 36,1195, 1991 in Japanese).

However, most of these factors exhibit a diversity of biologicalactivities other than participation in differentiation of blood cells invarious hemopoietic lineages including differentiation inmegakaryocyte-platelet lineages. For example, although IL-6 and IL-11actually exhibit in vivo thrombopoietic action, they stimulateproduction of acute phase protein, and in severe cases, cause cachexia.Moreover, IL-6 is accompanied with various clinical problems; forexample, it is possible for IL-6 to stimulate the growth of mesangiumcells in the kidney resulting in renal failure (Tadashi Matsuda et al.,Tanpakushitsu Kakusan Koso, 36, 1184, 1991 in Japanese). In addition,since IL-6 does not exhibit a high blood level during a thrombcytopenicphase, it is not considered as a physiological factor.

Platelets play an important role in a hemostatic mechanism. Diseasesinvolving decrease of platelets (Fanconi's syndrome, megakaryocyticthrombocytopenia, aplastic anemia, and the like) are clinicallydangerous, and in particular hemorrhaging cannot be controlled.Therefore, it is considered that isolation and identification of afactor which stimulates production of platelets is useful to prevent theabove-mentioned danger.

Currently, bone marrow transplantation is becoming a powerfultherapeutic means for treating leukenia etc., and the ratio ofsuccessful cases is increasing through the use of cytokines such aserythropoietin (EPO), granulocyte colony stimulating factor (G-CSF) etc.At present a problem in the bone marrow transplantation is a decrease inthe number of platelets, and if a thrombopoietic factor is available, itis expected that the ratio of successful cases will increase and aperiod of hospitalization will be shortened. Not only hemopoieticdiseases but also thrombocytopenia in chemotherapy and radio isotopictherapy of cancers may be controlled by thrombopoietin.

The present inventors, considering the various above-mentioneddifficulties with known factors, carried out various research to find afactor which stimulates production of platelets and is effective fortreatment of patients having thrombocytopenia or insufficient plateletfunction, and as a result, the present inventors found a novel factorwhich stimulates differentiation of megakaryocytes, cloned a gene codingfor said factor, constructed an expression vector, and succeeded inexpressing the gene to produce said factor.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a megakaryocytedifferentiation factor having the following properties;

(1) stimulating differentiation of megakaryocytes;

(2) exhibiting a molecular weight of 55 to 57 kD as determined by gelfiltration and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), andhaving no intermolecular disulfide linkage;

(3) exhibiting an isoelectric point of 6.5±0.5; and

(4) having at least one of the amino acid sequences shown in SEQ ID NO:1 to 9 in the Sequence Listing.

The present invention also provides a gene coding for the megakaryocytedifferentiation factor.

The present invention further provides an expression vector comprisingthe gene coding for the megakaryocyte differentiation factor.

The present invention moreover provides a host transformed with theexpression vector.

The present invention still further provides a process for production ofthe megakaryocyte differentiation factor using said host.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows an elution profile of protein (A280 --) and amegakaryocyte differentiation factor (acetylcholine esterase activity-∘-) from a Q-Sepharose column, wherein the elution was carried out byNaCl concentration gradient (0 to 1.0M) to obtain fractions 1 to 120.

FIG. 2 shows an elution profile of protein (A280 --) and amegakaryocyte differentiation factor (acetylcholine esterase activity-∘-) from a Phenyl-Sepharose column, wherein the elution was carried outby ammonium sulfate concentration gradient (30 to 0%) and ethyleneglycolconcentration gradient (0 to 50%) to obtain fractions Nos. 1 to 100,followed by 50% ethyleneglycol to obtain fraction Nos. 101 to 120.

FIG. 3 shows an elution profile of protein (A280 --) and amegakaryocyte differentiation factor (acetylcholine esterase activity-∘-) from an S-Sepharose column, wherein the elution was carried out byNaCl concentration gradient (0 to 0.5M) to obtain fractions Nos. 1 to100.

FIG. 4 shows an elution profile of protein (A280 --) and megakaryocytedifferentiation factor (acetylcholine esterase activity -∘-) from aHiroad 26/60 Superdex 75 Pg column, and a result of analysis of theresulting fractions by SDS-PAGE (lower part of the Figure).

FIG. 5 is an electrophoretic pattern showing a result of SDS-PAGEanalysis of the purified megakaryocyte differentiation factor.

FIG. 6 shows a result of an isoelectric focusing of the purifiedmegakaryocyte differentiation factor.

FIG. 7 is an electrophoretic pattern showing a result of sugar chainanalysis for the purified megakaryocyte differentiation factor bySDS-PAGE, wherein the column 1 shows a result for a non-treatedmegakaryocyte differentiation factor, and the column 2 shows a resultfor an endoglycosidase F-treated megakaryocyte differentiation factor(note, the band near the 35 kD position is derived from the enzymepreparation).

FIG. 8 is a graph comparing acetylcholine esterase activity ofmegakaryocytes derived from mouse bone marrow cells cultured for 5 daysin the presence or absence of purified megakaryocyte differentiationfactor (55 kDa protein) and with or without addition of IL-3.

FIG. 9A shows a result of acetylcholine esterase staining of mouse bonemarrow cells cultured for 4 days after the addition of IL-3 in theabsence of purified megakarocyte differentiation factor.

FIG. 9B shows a result of acetylcholine esterase staining of mouse bonemarrow cells cultured for 4 days after the addition of IL-3 in thepresence of purified megakarocyte differentiation factor.

FIG. 10A shows the result of May-Gruenwald-Giemsa's staining of mousebone marrow cells cultured for 4 days after the addition of IL-3 in theabsence of purified megakarocyte differentiation factor.

FIG. 10B shows the result of May-Gruenwald-Giemsa's staining of mousebone marrow cells cultured for 4 days after the addition of IL-3 in thepresence of purified megakarocyte differentiation factor.

FIG. 11 shows detection of RNA with a DNA probe (PCR product amplifiedwith KY100 and N1065 described in Example 2.1) wherein the RNA wasextracted from cultured Bombyx moli cells infected with recombinantvirus (TPO55-Bm NPV) comprising a gene coding for megakaryocytedifferentiation factor (Lane B), cells infected with wild type virus(B6E) (Lane A) or from non-transformed cells (Lane C).

FIG. 12 is a graph showing an expression of megakaryocytedifferentiation factor (TP55) in hemolymph of Bombyx mori afterseparation by Matrex Blue A column chromatography.

DETAILED DESCRIPTION

As starting materials for isolating the present megakaryocytedifferentiation factor, human cells, such as human cancer cells,preferably human epidermoid carcinoma cells A431, particularlypreferably human epidermoid carcinoma A431 grown in a protein-freemedium can be mentioned.

In addition to the above-defined megakaryocyte differentiation factor,the present invention relates to megakaryocyte differentiation factorsobtainable from transformants such as cells or animals constructed bygene technology and having the same amino acid sequence as theabove-defined megakaryocyte differentiation factor, an amino acidsequence wherein a portion of the above-defined megakaryocytedifferentiation factor is deleted, an amino acid sequence wherein aportion of the above-defined megakaryocyte differentiation factor isreplaced with other amino acid or amino acid sequence, or an amino acidsequence wherein one or more than one amino acid sequence is added tothe above-defined megakaryocyte differentiation factor, or having anamino acid sequence including a combination of said modifications.

Moreover, the present invention relates to megakaryocyte differentiationfactor having the amino acid sequence shown in SEQ ID NO: 30, an aminoacid sequence wherein a portion of the amino acid sequence shown in SEQID NO: 30 is deleted, an amino acid sequence wherein a portion of theamino acid sequence shown in SEQ ID NO: 30 is replaced with anotheramino acid or amino acid sequence, or an amino acid sequence wherein oneor more than one amino acid sequence is added to the amino acid sequenceshown in SEQ ID NO: 30, or having an amino acid sequence including acombination of said modifications.

The present invention also relates to genes coding for theabove-mentioned megakaryocyte differentiation factors. The presentinvention further relates a process for production of the megakaryocytedifferentiation factors using the gene by means of gene recombinationtechnology. The gene recombination technology follows conventionalprocedures by using a synthetic or natural polynucleotide coding for theamino acid sequence of the native megakaryocyte differentiation factor,an amino acid sequence wherein a portion of the native amino acidsequence is deleted, an amino acid sequence wherein a portion of thenative amino acid sequence is replaced with other amino acid or aminoacid sequence, or an amino acid sequence wherein one or more than oneamino acid is added to the native amino acid sequence, or coding for anamino acid sequence including a combination of said modifications, butnot limited to the above.

The above-mentioned various modifications can be carried out by aconventional technique such as site-specific mutagenesis.

The number of amino acids involved in the modification such as addition,deletion or replacement, is not limited. For addition, the number ofamino acid depends on the number of amino acids, for example, that ofthe functional peptide used in a hybrid protein with the megakaryocytedifferentiation factor of the present invention or that of a signalpeptide added to the present factor, namely depends on purpose of themodification. For deletion, the number of amino acids may be designed ordetermined so as to maintain megakaryocyte differentiation activity andit is, for example, 1 to 30, preferably 1 to 20 or it can be that ofregion other than the active region of the present factor. Forreplacement, the number of amino acids also may be designed ordetermined so as to maintain megakaryocyte differentiation activity andit is, for example, 1 to 10, preferably, 1 to 5.

An addition or improvement of signal sequence, choice of host-vectorsystem, and improvement of expression regulatory region may provideefficient expression. In addition, a host may be chosen to provide aglycosylated product. Moreover, a polynucleotide coding for at least oneof the amino acid sequences shown in SEQ ID NO: 1 to 9 may be used as aDNA probe for cloning a gene.

The present invention further provides a pharmaceutical compositioncomprising a megakaryocyte differentiation factor as an effectiveingredient. The pharmaceutical composition is preferably used as amedicament for thrombocytopenia.

In addition, the present megakaryocyte differentiation factors can beused to obtain specific antibodies according to a known procedure.

Now, the present invention is explained in more detail.

(1) Starting material

As a starting material for obtaining a novel protein of the presentinvention, a culture supernatant of cells derived from human epidermoidcarcinoma cell A431 (ATCC CRL 1555), rendered to be capable of growingin a protein-free medium according to Yamaguchi et al. method (YamaguchiN. et al., Cancer Res. 50, 7008, 1991), is mentioned. This cell line wasdesignated as Human epidermoid carcinoma SBM 330, and deposited as FERMBP-3911 with the Fermentation Research Institute Agency of IndustrialScience and Technology, 1-3, Higashi 1-chome, Tsukuba-shi Ibraki-ken(307) Japan, on Jul. 1, 1992.

(2) Assay method for megakaryocyte differentiation factor

To assay a megakaryocyte differentiation factor, megakaryocyte-seriescell lines (for example, CMK cells or cells derived therefrom), or mousebone marrow cells may be used. For example, activity of acetylcholineesterase which is known to be specifically detected in murinemegakaryocytes is carried out using mouse bone marrow cells according tothe Ishibashi et al. method (Ishibashi, T. et al., Proc. Natl. Acad.Sci. USA 86, 5953, 1989). In addition, histochemical detection ofmegakaryocytes is carried out by subjecting cultured bone marrow cellsto acetylcholine esterase staining and May-Gruenwald-Gremsa's stainingand the observing the shape of the stained cells.

(3) Purification of megakaryocyte differentiation factor

The megakaryocyte differentiation factor can be purified, for example,starting from a culture supernatant of A431 cells cultured in aprotein-free medium, concentration by ultrafiltration, and columnchromatography using, for example, Matrex Blue A (Amicon), Q-Sepharose(Pharmacia), phenyl-Sepharose (Pharmacia), S-Sepharose (Pharmacia) andHiload 26/60 Superdex 75 (Pharmacia) alone or in combination. Protein ismonitored by measuring A280 nm.

(4) Determination of partial amino acid sequence of megakaryocytedifferentiation factor

To determine an amino acid sequence, the megakaryocyte differentiationfactor purified in the section (3) is digested into fragments with aprotease such as Achromobacter Protease I (API) at 37° C. for 2 hours.The resulting peptide fragments are separated and recovered by reversephase HPLC (acetonitrile gradient in 0.1% trifluoroacetic acid) usingYMC-Pack AM-303 column. The peptide fragments thus obtained aresubjected to a sequencer such as a gas-phase sequencer obtained from,for example, Applied Biosystem. A definite purification process anddetailed properties of the megakaryocyte differentiation factor aredescribed in Example 1.

The present invention also provides gene coding for megakaryocytedifferentiation factor. The gene may be cDNA prepared from mRNA, genomicDNA, and synthetic DNA. For example, cDNA can be cloned by a polymerasechain reaction (PCR) using DNA (nucleotide) primers designed on thebasis of partial amino acid sequences as shown in Example 1 of amegakaryocyte differentiation factor purified from the above-mentionedhuman cells, such as human epidermoid carcinoma cells, for example A431cells. The cloning is described in detail in Examples 1 and 2.

The gene of the present invention further includes DNAs coding forprotein or glycoprotein having megakaryocyte differentiation activityand hybridizing with the nucleotide sequence of SEQ ID NO: 30.Nucleotide sequence of DNA cloned in Example 2 and an amino acidsequence predicted from the nucleotide sequence are shown in SEQ ID NO:30.

Thus once an amino acid sequence is determined, various mutatedmegakaryocyte differentiation factors, such as a polypeptide, whereinone or more than one amino acid is added to the native amino acidsequence or the amino acid sequence shown in SEQ ID NO: 30 andmaintaining megakaryocyte differentiation factor activity, a polypeptidewherein one or more than one amino acid is deleted from the native aminoacid sequence or the amino acid sequence shown in SEQ ID NO: 30 andmaintaining megakaryocyte differentiation factor activity, a polypeptidewherein one ore more than one amino acid is replaced with one or morethan one other amino acid, in the native amino acid sequecne or theamino acid sequence shown in SEQ ID NO: 30, and maintainingmegakaryocyte differentiation factor activity, or a polypeptideincluding a combination of the above-mentioned modifications such asaddition, deletion and/or replacement of amino acids maintainingmegakaryocyte differentiation factor activity can be designed andproduced.

According to the present invention, although the nucleotide sequenceshown in SEQ ID NO: 30 is disclosed, gene coding for the presentmegakaryocyte differentiation factor is not limited thereto. Once anamino acid sequence of the present native megakaryocyte differentiationfactor or an amino acid sequence of a mutated megakaryocytedifferentiation factor is determined, according to the degeneracy ofgenetic code, various nucleotide sequences coding for the same aminoacid sequence can be designed and prepared. In this case, codons usedwith a high frequency in a chosen host are preferably used.

A gene coding for the present megakaryocyte differentiation factor canbe obtained as cDNA according to Example 2, but the gene is not limitedto cDNA. Namely, once a nucleotide sequence coding for an amino acidsequence of native megakaryocyte differentiation factor is determined, agene coding for the native megakaryocyte differentiation factor can becloned as a cDNA according to a strategy different from the strategydefinitely described herein, and moreover a desired gene can be clonedfrom the genome of a cell producing the megakaryocyte differentiationfactor.

Where a desired gene is cloned from the genome, various primernucleotides or probe nucleotides used in Example 2 can be used as probesfor screening genomic DNA fragments. Moreover, other probes designed onthe basis of the nucleotide sequence described in SEQ ID NO: 30 can beused. General procedures for cloning a desired DNA from a genome arewell known in the art (see Current Protocols In Molecular Biology, JohnWiley & Sons, Chapters 5 and 6).

Gene coding for the native megakaryocyte differentiation factor of thepresent invention can also be prepared by chemical synthesis. It is easyin the art to chemically synthesize DNA using an automated DNAsynthesizer, for example, Applied Biosystems 396 DNA/RNA synthesizer.Accordingly a person with ordinary skill in the art can easilysynthesize DNA having the nucleotide sequence shown in SEQ ID NO: 30.

A gene coding for the present native megakaryocyte differentiationfactor using codons different from native codons, and a gene coding fora mutated megacaryocyte differentiation factor can be prepared bychemical synthesis as described above. Alternatively they can beobtained by site-specific mutagenesis using as a templete a DNA or RNAhaving a nucleotide sequence shown in SEQ ID NO: 30 together withmutagenic primers (for example, see, Current Protocols In MolecularBiology, John Wiley & Sons, Chapter 8).

Once a gene coding for the present megakaryocyte differentiation factoris obtained, the gene can be used to produce a recombinant megakaryocytedifferentiation factor according to a conventional gene recombinationtechnology. Namely, a DNA coding for the present megakaryocytedifferentiation factor is inserted into an appropriate expressionvector, the vector is introduced to appropriate host cells, thetransformed host cells are cultured, and the target megakaryocytedifferentiation factor is recovered from the culture (cells or medium).The present megakaryocyte differentiation factor can be biochemically orchemically modified, for example, N-terminal acylated.

In addition, on the basis of the nucleotide sequence shown in SEQ ID NO:30, a protein data base was searched with fasta Program (GCG package).As a result, the megakaryocyte differentiation factor belongs to thesuper family of serine protease inhibitor. On the other hand, in humanleucocyte elastase inhibitor, chicken ovalbumin Y gene product, humanplasminagen activator inhibitor 2 and human squamous cell carcinomaantigen, which are similar to the present megakaryocyte differentiationfactor in their expected steric structure, and distribution ofhydrophobic and hydrophilic amino acids, the N-terminal portion is notcleaved and forms a signal peptide. Accordingly, there is a possibilitythat in the present megakaryocyte differentiation factor, the N-terminalportion may function as a signal peptide and the megakaryocytedifferentiation factor may be secreted without cleavage of the signalpeptide. Further, the present megakaryocyte differentiation factor maybe modified so that the first methionine is deleted and the secondalanine is acetylated.

As hosts, both the prokaryote and enkaryote can be used. As prokaryote,bacteria such as Escherichia coli the genus Bacillus, for example, B.subtilis and the like can be used. As eukaryote, yeast such as the genusSaccharomyces, for example, S. serevisiae, insect cells such asSpodoptera frugiperda cells, Cabbage looper cells, Bombyx mori cells,animal cells such as human cells, monkey cells, mouse cells and the likecan be used. Moreover, insects per se, such as Bombyx mori may be used.

As expression vectors, plasmid, phage, phagemid, virus such as bacurovirus, vaccinia virus or the like can be used. A promoter in anexpression vector is selected depending on host used. For example, lacpromoter, trp promoter and the like can be used as bacterial promoters,and adhl promoter, pqk promoter and the like can be used as yeastpromoters. On the other hand, baculovirus polyhedrin promoter can beused as insect promoter, and Simian virus 40 early or late promoter canbe used for animal cells.

Transformation of a host with an expression vector can be carried outaccording to conventional procedures well known in the art, and theseprocedures are described in, for example, Current Protocols in MolecularBiology, John Wiley & Sons. Culturing of a transformant also can becarried out according to a conventional procedure.

Purification of a megakaryocyte differentiation factor from a culture oran insect body can be carried out according to conventional proceduresused for isolation and purification of a protein, for example,ultrafiltration, various types of column chromatography such asQ-Sepharose column chromatography and the like.

EXAMPLES

Next, the present invention is further explained by Examples.

Example 1

Purification of megakaryocyte differentiation factor

(1) Culturing of A431 cells

Frozen SBM 330 cells conditioned in a protein-free medium from A431cells were thawed, and cultured in a primary medium (Ham's F12 mediumcontaining 10% fetal bovine serum). Namely, the cells were plated in 10T flasks having a culture area of 150 cm², and cultured to confluence at37° C. in the presence of 5% CO₂. Next, the cells were peeled off with a0.25% trypsin solution (Chiba Kessei) and subcultured in 10 rollerbottles having a culture area of 850 cm² at 37° C. and 0.5 rpm for about3 days to recover 1.8×10⁹ cells. The cultured cells were attached to aceramic core (S-451) of an Opti-cell incubator (Charles River Inc.Wilmington, Mass.) and perfusion culturing was started using 10L of aprimary medium.

The perfusion culture was carried out at 37° C. with supplying oxygen at150 mmHg. The primary medium was replaced with a protein-free medium asfollow. Namely, after culturing for about 7 days in a primary medium, aprotein-free medium was supplied to the culture at a rate of 20L/day,and simultaneously the culture supernatant was recovered from theculture at the same rate. As a result, the primary medium containingserum was substantially completely replaced with the protein-free mediumby supplying about 100L of the protein-free medium. Thereafter, cellculture supernatant was continuously recovered to obtain 1000L of cellculture supernatant. A part (about 300L) of the cell culture supernatantthus obtained was concentrated to 2L using an ultrafiltration membrane(Milipore, Bedford, Mass.; MW 10,000 cut) and the concentrate wasdialyzed against 20 mM Tris/HCl buffer (pH 7.4).

(2) Assay of megakaryocyte differentiation factor using mouse bonemarrow cells

Myeloid cells were pushed out of the femur of female BDF₁ mouse andsuspended in an α-MEM medium (Flow Laboratories, Inc. McLean, Va., USA).Percoll layers having different densities (Pharmacia LKB Biotechnology,Tokyo) were overlaid, and the bone marrow cell suspension was putthereon, followed by centrifugation at 400 x g for 20 minutes.Mononuclear cells collected at the interface of a layer having a densityof 1.07 g/ml and a layer having a density of 1.08 g/ml were recoveredand washed once with A-MEM containing 10% FBS, and resuspended in thesame medium containing 0.5 mM diisopropylfluorophosphate. The suspensionwas then put into a plastic cell culture dish (Corning, N.Y., USA) andcultured at 37° C. in 5% carbon dioxide and 95% air for 2 hours. Duringthe culturing, at one hour from the start of culturing, the cell culturedish was replaced with a new one. After the culturing, cells were washedwith 10% FBS/α-MEM three times.

The non-adherent bone marrow mononuclear cells thus obtained weresuspended in 10% FBS/1% BSA/0.1 mM 2-mercaptoethanol/α-MEM, and platedin a 96-well microplate (Corning) in an amount of 5×10⁴ cell/well. Ifnecessary, to a test sample were added 25 U/ml mouse recombinant IL-3(Genzyme Corporation, Cambridge, Mass., USA) and 1 to 2 μg/ml anti-IL-6antibody (Boehringer Mannheim, Mannheim, FRG). Where anti-IL-6 antibodywas added, the test sample and the antibody were preincubated at 37° C.for an hour prior to seeding the cells.

The culturing was carried out at 37° C. in 5% CO₂ - 5% O₂ 90% N₂ for 4to 5 days. After culturing cells in each well of the microplate theywere washed twice with PBS, and lysed with 180 μl of 0.2% (w/v) TritonX-100, 1 mM EDTA, 0.12M NaCl, 50 mM HEPES (pH 7.5), and 20 μl of asubstrate, 5.6 mM acetylthiocholine iodide, was added thereon. Afterculturing with shaking at a room temperature for an hour, 20 μl of thesolution was transferred to a microplate for fluorescent assay (DynatechMicro FLUOR "B" Plate).

To the microplate were added 20 μl of 0.4 mm7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarine in acetonitrileand 160 μl of 0.2% (w/v) Triton X-100, 1 mM EDTA, 50 mM sodium acetate(pH 5.0), and fluorescent emission was measured by a fluorometer(excitation 365 nm, emission 450 nm).

For acetylcholine esterase staining, the cells were centrifuged by aCytospin to adhere the cells onto a slide glass, and fixed with 5%glutaraldehyde, 10 mM phosphate buffer (pH 6.7) for 15 minutes, and as asubstrate acetylthiocholine was used according to Mizoguchi's method(Method for Culturing Hemopoietic Stem Cells, Chugai Igaku, 1986, ed. Y.Miura, pp82-88). Namely, after fixing the cells, the slide glass waswashed with 0.1M phosphate buffer (pH 6.0), and to each slide glass waslayered a mixture of 1.5 ml of 0.67 mg/ml acetylthiocholine iodide, 0.1Mphosphate buffer (pH 6.0), 0.2 ml of 30 mM CuSO₄, 0.2 ml of 5 mMpotassium ferricyanide and 0.1 ml of 0.1M sodium citrate. The slideglass was incubated at a room temperature for 4 hours, and washed withwater. May-Gruenwald-Giemsa's staining is well known in hematology andwas carried out using reagents available from E. Merck (Darmstadt, FRG)wherein May-Gruenwald's staining was carried out for 4 minutes andGiemsa's staining was carried out for 10 minutes.

(3) Purification of megakaryocyte differentiation factor

A culture supernatant concentrate of A431 cells was dialyzed andcentrifuged to obtain a supernatant which was then applied to a MatrexBlue A Column equilibrated with 20 mM Tris/HCl buffer (pH 7.4), andafter washing the column with the same buffer, a bound fraction waseluted with the same buffer containing 2M NaCl. Megakaryocytedifferentiation activity detected by the above-mentioned method wasfound in the bound fraction. Therefore, the bound fraction was dialyzedagainst 20 mM Tris/HCl buffer (pH 7.4) and applied to a Q-Sepharosecolumn equilibrated with the same buffer. The column was thoroughlywashed and a megakaryocyte differentiation factor was eluted by NaClgradient (see FIG. 1). This factor was eluted near the position of 0.3to 0.5M NaCl.

The active fraction obtained from Q-Sepharose was made ammonium sulfate30% saturation, and was applied to a phenyl Sepharose columnequilibrated with 20 mM Tris/HCl buffer (pH 7.4) containing 30%saturation of ammonium sulfate. The megakaryocyte differentiation factorwas eluted by simultaneously forming concentration gradients of ammoniumsulfate (30% to 0%) and ethyleneglycol (0 to 50%) (see FIG. 2). Themegakaryocyte differentiation factor measured in the presence ofanti-IL-6 antibody was observed over several fractions in the beginningof the concentration gradient formation.

The fractions thus obtained were combined, thoroughly dialyzed against50 mM MES/NaOH buffer (pH 6.0), and applied to an S-Sepharose columnequilibrated with the same buffer. A bound fraction as eluted by a 0 to0.5M NaCl concentration gradient (see FIG. 3). Although activity waswidely distributed, relatively high activity was found in the beginningof the concentration gradient formation. The fractions obtained fromS-Sepharose were applied to a Hilord 26/60 Super Dex 75 (Pharmacia)column for gel filtration. The column used had be previouslyequilibrated with the same buffer, and elution was carried out with thesame buffer (see FIG. 4). Activity of megakaryocyte differentiationactivity was eluted near the position corresponding to a molecularweight of 55 to 57 kDa.

According to the above-mentioned steps, about 80 μg of a fractionshowing two bands near 55 to 57 kDa as analyzed by SDS-PAGE was obtainedfrom 300L of a culture supernatant of A431 derived cells (see FIG. 5).The two bands were correlated with the activity (FIG. 1). Accordingly,it was concluded that the two bands observed in said fraction correspondto a desired megakaryocyte differentiation factor.

(4) Properties of megakaryocyte differentiation factor

The present megakaryocyte differentiation factor has the followingproperties.

1) Molecular weight: about 55 kDa (gel filtration and SDS-PAGE) (FIGS. 4and 5)

The present factor exhibits two bands in SDS-PAGE, and there is nodifference in mobility between reducing condition and a non-reducingcondition. Therefore the factor does not have intermolecular disulfidelinkage.

2) Isoelectric point: 6.5±0.5 (FIG. 6)

A several bands are observed in the above-mentioned range.

3) The above-mentioned heterogeneity of the present factor can beexplained as heterogeneity in a sugar chain structure of glycoprotein.Namely, where the present factor is treated with endoglycosidase F whichis an asparagine linked sugar removing enzyme, the molecular weight ofthe present factor decreased to a molecular weight of about 40 kDa, andthe heterogeneity also decreased (FIG. 7), in SDS-PAGE. In addition,where a fraction exhibiting a single band and a fraction exhibiting twobands were digested with API and a peptide map was prepared byfractionation by a reversed phase HPLC, the difference was not observedbetween the two fractions.

4) The present factor contains at least one of the amino acid sequencesshown in SEQ ID NO: 1 to 9.

5) Biological activity

Where mouse bone marrow cells were cultured in the presence of apurified megakaryocyte differentiation factor and IL-3, theproliferation and differentiation of megakaryocytes are observed (FIGS.8, 9 and 10). FIG. 8 shows the result of measurement for acetylcholineesterase activity of megakaryocytes; FIG. 9 shows the result ofacetylcholine esterase staining of the cultured cells (x 20); and FIG.10 shows the result of May-Gruenwald-Giemsa's staining of the culturedcells (x 100). In both the FIGS. 9 and 10, it is seen thatmegakaryocytes increased in the presence of a megakaryocytedifferentiation factor (B) in comparison with in the absence of the same(A).

(5) Structure of megakaryocyte differentiation factor

To characterize the structure of a purified megakaryocytedifferentiation factor, the factor was digested with API and thestructures of the resulting fragments were determined. After thedigestion of the factor with API, each fragment was recovered byreversed phase HPLC, and the structures for appropriate fractions weredetermined. As a result, the peptide fragments had amino acid sequencesshown in SEQ ID NO: 1 to 9.

Example 2

Determination of Structure of CDNA for megakaryocyte differentiationfactor

1. Analysis of cDNA nucleotide sequence of megakaryocyte differentiationfactor by PCR (1)

Oligonucleotides NIO65 (SEQ ID NO: 10; corresponding to 449-486 of SEQID NO: 30) and NI067 (SEQ ID NO: 11; corresponding to 1049-1080 of SEQID NO: 30) were synthesized by designing nucleotide sequences on thebasis of the amino acid sequences shown in SEQ ID NO: 3 and 4respectively.

Total RNA was purified from A431 cells using ISOGEN (Wako Pure Chemical)according to the manufacturer's instructions. RNA having poly A waspurified from the total RNA, and a reaction was carried out using a3'-RACE Kit (Gibco BRL). Namely, the above-mentioned oligomer NI065 andoligomer 3'-RACE adaptor primer (SEQ ID NO: 12) attached to the 3'-RACEKit (Gibco BRL) were used to carry out polymerase chain reaction (PCR)according to instructions included with the kit.

The reaction product was then subjected to second PCR using the primerNI067 and the oligomer 3'-RACE adapter primer included in the 3'-RACEKit (Gibco BRL) to obtain a DNA fragment of about 900 base pairs. Next,using a direct nucleotide sequence determination method for a PCRproduct, according to U. Gyllensten et. al., Proc. Natl. Acad. Sci. USA85: 7652 (1988), the DNA fragment of about 900 base pairs was directlyused as a reaction substrate to determine a nucleotide sequence of aportion representing protein and a portion downstream of the proteinportion using a Taqu Dye Deoxy Terminator Cycle Sequencing kit availablefrom Applied Biosystem and a fluorescent nucleotide sequencer (AppliedBiosystem, Type 370A) according to the manufacturers instruction. As aresult, a sequence from nucleotide number 1081 to 1950 of SEQ ID NO: 30was shown.

On the basis of this sequence, oligomer KY100 (SEQ ID NO: 13;corresponding to 1255-1236 of SEQ ID NO: 30) was synthesized. Thereaction product obtained by the PCR using NI065 and the oligomer3'-RACE adapter primer attached to the 3'-RACE Kit (Gibco BRL) was usedas a reaction substrate to carry out a further PCR using NI065 andKY100. As a result, a DNA fragment of 807 base pairs was obtained.

This DNA fragment of 807 base pairs was directly used as a reactionsubstrate to determine its nucleotide sequence using Taq Dye DeoxyTerminator Cycle Sequencing kit available from Applied Biosystem and afluorescent nucleotide sequencer according to the manufacture'sinstruction. As a result, a nucleotide sequence from nucleotide number487 to 1080 of SEQ ID NO: 30 was shown. On the basis of this sequence,oligomers NI073 (SEQ ID NO: 14; corresponding to 864-886 of SEQ ID NO:30), NI074 (SEQ ID NO: 15; corresponding to 1012 to 992 of SEQ ID NO:30), and NI075 (SEQ ID No: 16; corresponding to 802-782 of SEQ ID NO:30) were synthesized.

2. Analysis of cDNA nucleotide sequence of megakaryocyte differentiationfactor by PCR (2)

A. Preparation of mRNA from megakaryocyte differentiation factorexpressing cell line (A431)

From 1.1 g of frozen cells of human epidermoid carcinoma cell line(A431), 25 μg of mRNA was extracted and purified using an RNA extractionkit and an mRNA purification kit available from Pharmacia-LKB.

B. Preparation of DNA phage library from megakaryocyte differentiationfactor expressing cell line (A431)

(1) Synthesis of cDNA

From 5 μg of the mRNA derived from A431, cDNA was synthesized using acDNA synthesis kit Time Saver available from Pharmacia-LKB. First 5 μgof mRNA dissolved in 20 μl of distilled water treated withdiethylpyrocarbonate (DEPC) was heated at 65° C. for 10 minutes andcooled on ice. 11 μl of a first strand reaction mixture, 1 μl of DTTsolution and 1 μl of 130 U/ml NotI/oligomer 18 primer solution(Pharmacia-LKB) were added thereto, and the mixture was incubated at 37°C. for an hour.

The reaction mixture was added to a second strand reaction mixture, andthe mixture was incubated at 12° C. for 30 minutes and 22° C. for anhour, and heated at 65° C. for 10 minutes. 100 μl of a mixture ofphenol/chloroform/isoanylalcahol (25:24:1; abbreviated as PChereinafter) was added thereto, and the mixture was vigorously stired,centrifuged at 14,000×9 for one minute to obtain a supernatant, whichwas then fractionated by a Sephacryl S-400 spin column (Pharmacia LKB)to obtain 100 μl of cDNA solution.

(2) Addition of EcoRI adaptor

To 100 μl of the CDNA solution were added 5 μl of 10 U/ml EcoRI adaptor(Pharmacia LKB), 30 μl of polyethylene glycol buffer, 1 μl of 1/5diluted ATP solution and 1 μl of T4 DNA ligase, and the mixture wasincubated at 37° C. for an hour. After heating at 65° C. for 10 minutes,1.5 μl of ATP solution and 1 μl of T4 polynucleotide kinase were addedthereto and the mixture was incubated at 37° C. for 30 minutes. Afterheating at 65° C. for 10 minutes, 2 μl of 20 U/μl NotI was added to themixture, which was then incubated at 37° C. for an hour. 150 μl of PCwas added to the mixture, which was then vigorously stirred andcentrifuged at 14,000 x g for a minute, and the supernatant wasfractionated on Sephadex S-400 spin column to obtain 150 μl of cDNAsolution.

(3) Incorporation of CDNA into phage vector and in vitro packaging

After digesting with EcoRI and NotI, to 15 μl of the cDNA solution wereadded 2 μg of dephosphorylated λgt11D (Pharmacia LKB). After ethanolprecipitation, the precipitate was dissolved in 8 μl of ligase buffersolution, and 1 μl of 1/75-diluted ATP solution and 1 μl of T4 DNAligase were added to the solution, which was then incubated at 16° C.for 30 minutes and stored on ice.

An in vitro packaging reaction was carried out using Gigapack II Gold(Stratagene), and a library of 3.22×10⁶ pfu of recombinant phages wasobtained from said 3 ligase reaction products. Said library wasamplified in an E. coli Y1090 r⁻ host to obtain 6.0×10¹⁰ pfu/ml of anA431 phage library stock.

C. Identification and isolation of cDNA fragment for megakaryocytedifferentiation factor by PCR

(1) Amplification of cDNA insert fragment in A431 phage library by PCR

10 μl of a stock solution of 6.0×10¹⁰ pfu/ml A431 phage library(corresponding to 6.0×10⁸ pfu) was used as a template DNA for PCRreaction, and 5 μl of 10 x PCR buffer, 8 μl of 1.25 mM 4 dNTPs, 2 μl of1 OD/ml λgt11-forward primer (λgt 11F) (SEQ ID NO: 17), 2 μl of 1 OD/mlλgt11-reverse primer (λgt 11R) (SEQ ID NO: 18), and 1 μl of 5 U/μl TaqDNA polymerase (Perkin Elmer Cetus) were added, and the total volume ofthe mixture was made to be 50 μl with DEPC-treated distilled water. 30reaction cycles of 93° C. for a minute, 55° C. for 2 minutes and 72° C.for 3 minutes were carried out, and the reaction mixture was incubatedat 72° C. for 10 minutes. As a result of an analysis by 1% Agarose gelelectrophoresis, a smeary pattern ranging 0.8 to 6 kb was shown.

(2) PCR analysis using cDNA insert DNA amplification fragment mixture asa template and using TP7 (SEQ ID NO: 20; corresponding to 683-703 of SEQID NO: 30), /TP10, TP7/TP6 (SEQ ID NO: 19; corresponding to 1036-1001 ofSEQ ID NO: 30), TP8 (SEQ ID NO: 21; corresponding to 941-964 of SEQ IDNO: 30), TP10 (SEQ ID NO: 22; corresponding to 1036-986 of SEQ ID NO:30) and TP8/TP6 as primers

1 μl of a 1/5000-diluted solution of the above-mentioned PCR reactionproduct was used as a template DNA for PCR reaction, and 5 μl of 10 xPCR buffer, 8 μl of 1.25 mM 4 dNTPs, 2 μl each of 1 OD/ml primers incombination as described hereinafter and 1 μl of Perfect Match(Stratagene) were added, and a total volume of the mixture was made to49 μl with DEPC-treated distilled water.

The reaction mixture was heated at 95° C. for 5 minutes and 60° C. for 5minutes, 1 μl of 5 U/μl Taq DNA polymerase (Perkin Elmer Cetus) wasadded thereon, and 30 cycles of 94° C. for a minute, 60° C. for 2minutes and 72° C. for 3 minutes were carried out, followed by anincubation at 72° C. for 10 minutes. As primers, TP7/TP10, TP7/TP6,TP8/TP10, and TP8/TP6 were used.

As a result of an analysis of the PCR reaction product by 2% agarose gelelectrophoresis, bands of 354 bp, 354 bp, 96 bp and 96 bp were obtainedcorresponding to primers respectively.

(3) Analysis for primary sequence of PCR amplification products (354 bp)obtained by using primers TP7/TP10 and TP7/TP6

The bands of said PCR amplification products (354 bp) obtained usingprimers TP7/TP10 and TP7/TP6 were excised from the 2% agarose gel afterthe electrophoresis, and to the excised agarose gel pieces was added 50μl of DEPC-treated distilled water, and the mixture was heated at 45° C.for 30 minutes. To 2 μl of this solution as a template DNA, were added 5μl of 10 x PCR buffer, 8 μl of 1.25 mM 4 dNTPs, 2 μl each of 1 OD/mlprimers in combination and 1 μl of Perfect Match (Stratagene), and atotal volume of the reaction mixture was made to 49 μl with DEPC-treateddistilled water. The mixture was heated at 95° C. for 5 minutes and 60°C. for 5 minutes, 1 μl of 5 U/μl Taq DNA polymerase (Perkin Elmer Cetus)was added, and 30 reaction cycles of 94° C. for a minutes, 60° C. for 2minutes and 72° C. 3 minutes were carried out, followed by incubation at72° C. for 10 minutes. As primes for the above-mentioned PCR reaction,TP7/TP10 and TP7/TP6 were used. Bands of the PCR reaction products (each354 bp) were excised from 2% agarose gel of the electrophoresis,extracted and purified, and the product was inserted into pCR II(Invitrogen), which was then used to transform E. coli INVαF'(Invitrogen). Plasmid DNA was extracted and purified, and it wasconfirmed by EcoRI digestion that DNA fragment of 354 bp had beeninserted.

Primary sequence of the DNA insert fragment was analyzed using M13forward primer (M13F) (SEQ ID NO: 23) and M13 reverse primer (Ml3R) (SEQID NO: 24) (Aplied Biosystem's automated sequencer, Model 370A). As aresult, a 296 bp sequence corresponding to the nucleotide number 704 to999 of SEQ ID NO: 30 was found, and this sequence contained C-terminal 3amino acids (XRK; but ERK from DNA nucleotide sequence) of SEQ ID NO: 9corresponding to the downstream portion of primer TP7, N-terminal 5amino acids (ADLSG) of SEQ ID NO: 6 corresponding to the upstreamportion of primer TP6, and 8 amino acids (YLRALGLK) of SEQ ID NO: 5corresponding to primer TP8, revealing that the PCR reaction products(each 354 bp) is a part of cDNA coding for megakaryocyte differentiationfactor.

3. Screening of cDNA coding for megakaryocyte differentiation factor

A. Preparation of cDNA plasmid library from megakaryocytedifferentiation factor expressing cell line (A431)

(1) Synthesis of first strand cDNA From 5 μg of mRNA derived from

A431 cell line, cDNA was synthesized using a Super Script plasmid systemavailable from GIBCO. First 2 μl of NotI primer adaptor was added to 5μg of mRNA dissolved in 5 μl of diethylpyrocarbonate (DEPC)-treateddistilled water, and the mixture was heated at 70° C. for 10 minutes andcooled on ice. 4 μl of 5 x first strand buffer, 2 μl of 0.1 M DTTsolution, 1 μl of 10 mM 4dNTPs and 1 μl of DEPC-treated distilled waterwere added thereto, and the mixture was incubated at 37° C. for 2minutes. 5 μl of a Super Script reverse transcriptase was added to thereaction mixture, which was then incubated at 37° C. for one hour andthen put on ice to stop the reaction.

(2) Synthesis of second strand CDNA

To 18 μl of 20 μl reaction mixture for the first strand cDNA synthesiswere added 93 μl of DEPC-treated distilled water, 30 μl of 5 x secondstrand buffer, 3 μl of 10 mM 4 NTPs, 1 μl of 10 U/μl E. coli DNA ligase,4 μl of 10 U/μl E. coli DNA polymerase and 1 μl of 2 U/μl E. coli RNaseH, and the mixture was incubated at 16° C. for 2 hours. 2 μl (10 U) ofT4 DNA polymerase was added to the reaction mixture, which was thenincubated at 16° C. for 5 minutes.

The reaction mixture was put on ice, and after adding 10 μl of 0.5M EDTAand 150 μl of PC thereto, was vigorously stirred and centrifuged at14,000 x g for 10 minutes, and 140 μl of the supernatant was transferredto a fresh centrifuge tube. 70 μl of 7.5M ammonium acetate and 0.5 ml ofethanol were added to the supernatant, which was then allowed to standat -80° C. for 30 minutes. The mixture was centrifuged at 14,000 x g for10 minutes, and after removing the supernatant, the precipitate waswashed with 0.5 ml of 70% ethanol and dried under a reduced pressure.

(3) Addition of BstXI adapter

The above-mentioned cDNA precipitate was dissolved in 25 μl ofDEPC-treated distilled water, 10 μl of 5 x T4 DNA ligase buffer, 10 μlof BstXI adapter (Invitrogen) and 5 μl of T4 DNA ligase were added tothe solution, which were then incubated at 16° C. for 16 hours. 50 μl ofPC was added to the mixture, which was the vigorously stirred, andcentrifuged at 14,000 x g for 5 minutes. 45 μl of the supernatant wastransferred to a fresh centrifuge tube. 25 μl of 7.5M ammonium acetateand 150 μl of ethanol were added to the tube, which was stirred andallowed to stand at -80° C. for 30 minutes. After centrifuging at 14,000x g for 10 minutes to remove supernatant, the precipitate was washedwith 0.5 ml of 70% ethanol and dried under a reduced pressure.

(4) NotI digestion

The above-mentioned cDNA precipitate was dissolved in 41 μl ofDEPC-treated distilled water, and 5 μl of REAct 7 buffer and 4 μl ofNotI were added to the solution, which was incubated at 37° C. for 2hours. 50 μl of PC was added to the mixture, which was then vigorouslystirred and centrifuged at 14,000 x g for 10 minutes, and 45 μl of thesupernatant was transferred into a centrifuge tube.

(5) Elimination of adapter and size fractionation of partial cDNA

The above-mentioned cDNA solution was fractionated using a Quick SpinColumn Linker 5 (Boehringer Mannheim). 50 μl of 40 μg/μl cDNA wasobtained.

(6) Incorporation of cDNA into phagevector and transformation of E. coli

To 37.5 μl of the above-mentioned cDNA solution were added 12.5 μl ofpCR/CMV (Invitrogen) vector (29 μg/μl) digested with Not I and BstXI,and further added were 400 μl of Takara Ligation kit A solution and 50μl of B solution, and the mixture was incubated at 16° C. for 30minutes, and 1 ml of Max Efficiency DH5a competent cells (BRL) weretransformed to obtain 71,550 recombinant clones. All colonies werecollected from plate (2.86×10⁷ cells/ml) and stored at -80° C. in thepresence of 20% glycerol.

B. Screening of megakaryocyte differentiation factor CDNA by colonyhybridization

Using the cDNA plasmid library derived from A431 cell line, a total of227,000 (3700/plate) colonies were formed on 60 plates of 9 cm diameter,and the colonies were replicated to nitrocellulose filters. A probe wasprepared by carrying out PCR (as described hereinbefore) using primer NI067 and 3'-RACE adaptor (GIBCO BRL) to obtain a 900 bp PCR product,digesting the PCR product with Bam HI to obtain two DNA fragments (0.5kb and 0.4 kb) and nick-translating the DNA fragments with [α-³ P]dCTP.

For the colony hybridization, the filter was incubated in 5 x SSC, 25 mMphosphate buffer (pH 7.4), 5 x Denhaldt's solution, 1% SDS, 100 μg/mlheat denatured salmon sperm DNA and 50% formamide at 42° C. for 18hours, and washed with 5 x SSC, 0.1% SDS at 40° C. for 20 minutes and45° C. for 20 minutes. Detection was carried out by exposing BAS 2000(Fuji Film) for 18 hours.

First, second and third screening was carried out to obtain 4 clones,i.e., TP290, TP308, TP310 and TP317. The length of insert cDNA was 1.2kb, 1.1 kb, 1.2 kb and 1.2 kb respectively. The TP290, TP310 and TP317cover a region downstream from the nucleotide number 685 of SEQ ID NO:30.

4. Analysis of cDNA nucleotide sequence coding for megakaryocytedifferentiation factor by PCR (3)

A. Preparation of mRNA from megakaryocyte differentiation factorexpressing cell line (HPC-Y11)

From 1.1 g of frozen cells of human pancreatic cancer cell line(HPC-Y11), 50 μg of mRNA was extracted and purified using an RNAextraction kit and mRNA purification kit available from Pharmacia-LKB.

B. Preparation of CDNA phage library from megakaryocyte differentiationfactor expressing cell line (HPC-Y11)

(1) Synthesis of cDNA

From 5 μg of mRNA derived from HPC-Y11, CDNA was synthesized using aPharmacia-LKB's Time Saver CDNA synthesis kit. First, 5 μg of mRNA wasdissolved 20 μl of diethylpyrocarbonate (DEPC)-treated distilled water,and the solution was heated at 65° C. for 10 minutes and cooled on ice.11 μl of first strand reaction mixture, 1 μl of DTT solution and 1 μl ofNotI/oligomer 18 primer solution were added to the mixture, which wasthen incubated at 37° C. for an hour.

100 μl of second strand reaction mixture was added to the mixture, whichwas then incubated at 12° C. for 30 minutes and at 22° C. for an hour,and heated at 65° C. for 10 minutes. 100 μl of phenol-chloroform-isoamyl alcohol (25:24:1, abbreviated as PC) was added to themixture, which was then vigorously stirred and centrifuged at 14,000 x gfor a minute, and the supernatant was fractionated using a SephacrylS-400 spin column (Pharmacia-LKB) to obtain 100 μl of CDNA solution.

(2) Addition of EcoRI adaptor

To 100 μl of the cDNA solution, were added 5 μl of EcoRI adapter(Pharmacia-LKB), 30 p1 of polyethylene glycol, 1 μl of ATP solution and1 μl of T4 DNA ligase, and the mixture was incubated at 37° C. for anhour. After heating at 65° C. for 10 minutes, 1.5 μl of ATP solution and1 μl of T4 polynucleotide kinase were added to the mixture, which wasthen incubated at 37° C. for 30 minutes. After heating at 65° C. for 10minutes, 12 μl of Not I was added to the mixture, which was thenincubated at 37° C. for an hour. 150 μl of PC was added to the mixture,which was then vigorously stired and centrifuged at 14,000 x g for aminute, and the supernatant was fractionated using a Sephacryl S-400spin column to obtain 150 μl of CDNA solution.

(3) Incorporation of CDNA into phage vector and in vitro packaging

To 15 μl of the cDNA solution, was added 2 μg of λgt 11D (Pharmacia-LKB)which had been digested with EcoRI and Not I and dephosphorydated, andafter ethanol precipitation, the precipitate was dissolved in 8 μl ofligase buffer. 1 μl of 1/75-diluted ATP solution and 1 μl of T4 DNAligase were added to the mixture, which was then incubated at 16° C. for30 minutes and stored on ice. An in vitro packaging reaction was carriedout using a Giga Pack II Gold (Strotagene), and 5.34×10⁶ pfu ofrecombinant phages were obtained from the above-mentioned 3 ligasereaction products. The library was amplified in E. coli Y1090 r⁻¹ hostto obtain a stock of 1.7×10¹¹ pfu/ml HPC-Y11 phage library.

C. Identification and isolation of 5'-portion of megakaryocytedifferentiation factor CDNA

(1) Amplification by PCR of HPC-Y11 phage library cDNA insert DNAfragment primer NI074 upstream portion.

To 1 μl (corresponding to 1.7×10⁹ pfu) of 6.0×10¹⁰ pfu/ml HPC-Y11 phagelibrary stock solution as a template DNA for PCR reaction, were added 5μl of 10 x PCR buffer, 8 μl of 1.25 mM 4 dNTPs, 1 μl of 10OD/mlλgt11-forward F1 primer (SEQ ID NO: 25), 1 μl of 50 D/ml NI074primer and 1 μl of Perfect Match (Stratagene), and a total volume wasmade to be 49 μl with DEPC-treated distilled water.

After heating the reaction mixture at 95° C. for 5 minutes and 60° C.for 5 minutes, 1 μl of 5 U/μl Taq DNA polymerase (Perkin Elmer Cetus)was added thereto, and 35 reaction cycles of 94° C. for a minute, 60° C.for a minute and 72° C. for 2 minutes were carried out, followed byincubation at 72° C. for 10 minutes. A result of analysis by 2% agarosegel electrophoresis showed a smeary pattern ranging from 0.3 to 6 kb.

(2) PCR analysis using a template which is a PCR amplification fragmentmixture prepared by using λgt11F1/NI074 primer, and using as primersλgt11F2 (SEQ ID NO: 26)/NI075, λgt11F2/TP12 (SEQ ID NO: 28;corresponding to 703-683 of SEQ ID NO: 30), λgt11F2/TP11 (SEQ ID NO: 27;corresponding to 619-599 of SEQ ID NO: 30), λgt11F2/TP13 (SEQ ID NO: 29;corresponding to 595-575 of SEQ ID NO: 30), TP7/NI074, TP7/NI075, andNI073/NI074

1 μl of a 1/100 diluted solution of the above-mentioned PCR reactionproduct was used for a template DNA for PCR reaction, and 5 μl of 10 xPCR buffer, 8 μl of 1.25 mM 4 dNTPs, 0.5 μl each of 10 OD/ml primers incombination as described hereinafter, and 1 μl of Perfect Match(Stratagene) were added thereto, and a total of the reaction mixture wasmade to be 49 μl with DEPC-treated distilled water. The reaction mixturewas heated at 95° C. for 5 minutes and at 60° C. for 5 minutes, 1 μl of5 U/μl Taq DNA polymerase (Perkin Elmer Cetus) was added thereto, and 35reaction cycles of 94° C. for a minute, 60° C. for 2 minutes and 72° C.for 2 minutes were carried out, followed by an incubation at 72° C. for10 minutes.

As primers, λgt11F2/NI075, λgt11F2/TP12, λgt11F2/TP11, λgt11F2/TP13,TP7/NI074, TP7/NI075, and NI073/NI074 were used. As a result of 2%agarose gel electrophoresis, bands of 969 bp, 870 bp, 786 bp, 762 bp,330 bp, 120 bp and 149 bp were obtained corresponding to the primers.

(3) PCR analysis and primary sequence analysis using λgt11F/TP11 andλgt11F/TP13 primer, for PCR amplification product (969 bp) preparedusing F2/NI075 primers

0.5 μl of the above mentioned PCR reaction product (969 bp) prepared byusing λgt11F2/NI075 as primers was used as a template DNA for PCRreaction, and 5 μl of 10 x PCR buffer, 8 μl of 1.25 mM 4 dNTPs, 1 μl of10 OD/ml λgt11F primer, 1 μl of 10 OD/ml TP11 primers or 1 μl of 10OD/ml TP13 primers, and 1 μl of Perfect Match (Stratagene) were added,and the total volume was made to be 49 μl with DEPC-treated distilledwater.

The reaction mixture was heated at 95° C. for 5 minutes and 60° C. for 5minutes, 1 μl of 5 U/μl Taq DNA polymerase (Perkin Elmer Setus) wasadded thereto, and 35 reaction cycles of 94° C. for a minute, 60° C. for2 minutes and 72° C. for 2 minutes were carried out, followed by anincubation at 72° C. for 10 minutes.

Bands of PCR reaction products (678 bp and 654 bp, respectively) wereexcised from 2% agarose gel after electrophoresis, extracted andpurified, and inserted into PCRII (Invitrogen), which was then used totransform E. coli IN VαF' (Introgen). Plasmid DNA was extracted andpurified from the transformant, and digested with EcoRI to confirm thata 0.7 kb DNA fragment was inserted. The primary sequence of the insertedDNA fragment was analyzed by using M13 forward primer M13F and M13reverse primer M13R (Applied Biosystems automated sequencer Model 370A).

As a result, a sequence of 619 bp corresponding to nucleotide number 1to 619 of SEQ ID NO: 30 was found, and the nucleotide sequence of 133nucleotides consisting of nucleotide number 487 to 619 of SEQ ID NO: 30conformed to the N-terminus of the primary sequence found in Example2.1. In this 619 bp sequence there are 19 amino acids of SEQ ID NO: 3(VERVDFTNHLEDTR RNINK from DNA nucleotide sequence) and 5 amino acids(LYDAK) of SEQ ID NO: 7, and it was clarified that this PCR reactionproducts (each 0.7 kb) was a part of CDNA coding for megakaryocytedifferentiation factor.

It was considered that the translation starting methionine correspondsto 74th nucleotide, and 5'-non translational region consisted of 73 bp.Accordingly, it was clarified that these PCR reaction products (each 0.7kb) contain N-terminus of structural gene for megakaryocytedifferentiation factor.

5. Analysis of cDNA nucleotide sequence coding for megakaryocytedifferentiations factor by PCR (4)

Among an N-terminal portion of structural gene for a megakaryocytedifferentiation factor derived from HPC-Y11 and a sequence considered tobe a 5'-non-translational region, obtained in the section C(3), thesequence of nucleotide numbers 12 to 31 of SEQ ID NO: 30 which is asequence part considered to be a 5'-non-translational region was used asa basis to synthesize an oligomer NI 083 (SEQ ID NO: 31).

The RNA having poly A prepared from A431 cells in the section 1., aPreamplification System (Gibco BRL) and random hexamers attached to theSystem were used to synthesize first strand cDNA according to includedinstructions, and PCR was carried out by using NI083 and NI074 as wellas Ampli Taq (Takara). As a result, a DNA fragment of 1001 bp which is acDNA fragment for a megakaryocyte differentiation factor was obtained.

This DNA fragment, a PCR product, of 1001 bp was directly used as asubstrate for sequencing on a Taq Dye Deoxy Terminator Cycle Sequencingkit (Applied Biosystem) and a fluorescent sequencer (Applied BiosystemType 370A) according to the included manufacturers instruction. As aresult, a sequence of nucleotide numbers 32-486 of SEQ ID NO: 30 wasfound. In addition, a result obtained for nucleotide numbers 487 to 991of SEQ ID NO: 30 conformed to the sequence obtained in the section 1.

By combining with the sequence obtained in the section 1., thenucleotide sequence 32-1950 of SEQ ID NO: 30 which is cDNA nucleotidesequence coding for a megakaryocyte differentiation factor of A431 cellwas determined.

For this nucleotide sequence, all of possible 3 reading frames weremechanically translated into amino acid sequences and it was found thatone of them has a region which can be translated to a continuing aminoacid sequence containing all the amino acid sequences shown in SEQ IDNO: 1 to 9, and the reading frame of the megakaryocyte differentiationfactor was determined.

It was found that in this reading frame a codon for methionine(nucleotide numbers 74 to 76) found at a translation start positionexists, and from this position a region which can be translated to anamino acid sequence containing the amino acid sequences shown in SEQ IDNO: 1 to 9 continues up to the nucleotide position 1213 of SEQ ID NO:30, and it was determined that the nucleotide sequence from the position74 to the position 1213 of SEQ ID NO: 30 is the region translated tomegakaryocyte differentiation factor.

The nucleotide sequence GCAATGG (nucleotide numbers 71 to 77 of SEQ IDNO: 30) encompassing the methionine codon of nucleotide numbers 74 to 76of SEQ ID NO: 30 corresponds to a sequence (G/A-N-N-A-T-G-G)encompassing a methionine codon frequently present at a translationstart site found by M. Kozak, Nucleic Acids Research (1981) Vol. 9,p5233-5252.

Primary sequence of megakaryocyte differentiation factor was thusclarified, the number of amino acids was 380 in the structural gene, anexpected molecular weight was 42904.43, and an expected isoelectricpoint was 6.79. SEQ ID NOS: 1-6 and 9 give the partial amino acidsequences of peptide fragments recovered by reversed phase HPLCfollowing digestion. After cloning the DNA sequence, the completesequences of these peptides were determined to be that of amino acids188-196, 181-197, 126-144, 325-341, 289-297, 305-324, and 204-213,respectively, of SEQ ID NO: 34. SEQ ID NOS: 7 and 8 correspond to aminoacids 121-125 and 284-288, respectively, of SEQ ID NO: 34. In addition,A poly A addition signal AATAAA sequence is present at nucleotidenumbers 1933 to 1998 of SEQ ID NO: 30.

Example 3

Isolation and identification of CDNA coding for megakaryocytedifferentiation factor from A431 by PCR and construction of expressionvector

Oligomers NI078 (SEQ ID NO: 32) and NI079 (SEQ ID NO: 33) weresynthesized on the basis of the sequence (SEQ ID NO: 30) obtained inExample 2. Note in the NI078, the sequence of nucleotide numbers 13 to37 encompassing the translation start methionine codon conforms to thesequence of the nucleotide numbers 74 to 98 of SEQ ID NO: 30 and anEcoRI recognizing site (nucleotide numbers 4 to 9) and an Nru Irecognizing site (nucleotide numbers 8 to 13) were artificially added;and in the NI079, the sequence of the nucleotide numbers 17 to 49conforms to the nucleotide sequence of the nucleotide numbers 1237 to1269 of SEQ ID NO: 30, and an EcoRI recognizing site (nucleotide numbers3 to 8) and an Not I recognizing site (nucleotide numbers 9 to 16) wereartificially added.

The RNA having poly A prepared from A431 cells in Example 2 section 1.,a Preamplification System (Gibco BRL), and Oligomers, i.e., randomhexamers included in the system were used according to the includedmanufacturer's instructions to synthesize first strand CDNA, and PCR wascarried out using the synthesized DNA as a template and NI078 and NI079as primers and using Ampli Taq (Perkin Elmer Cetus). As a result, a DNAfragment of 1224 base pairs which is a cDNA fragment for a megakaryocytedifferentiation factor and has all information relating to megakaryocytedifferentiation factor, was obtained.

This DNA fragment was treated with EcoRI to generate EcoRI cohesivesites at both the ends of the cDNA coding for a megakaryocytedifferentiation factor in virtue of EcoRI recognizing sites artificiallyadded to the oligomers NI078 (SEQ ID NO: 32) and NI079 (SEQ ID NO: 33).This cDNA fragment coding for megakaryocyte differentiation factor wasintroduced into a mammalian expression vector pdKCR-DHFD at its EcoRIrecognizing site to obtain pdKCR-DHFR-TPO55.

The animal cell expression vector pdKCR-dhfr (Oikawa, S. et. al.,Biochem. Biophys. Res. Commun. 164, 39, 1989) is a derivative of pKCR(O'Hare et. al., Pro. Natl. Acod. Sci. USA, 78, 1527, 1981) and has SV40early promoter and a rabbit β-globin gene and dhfr (dehydrofolatereductase) gene. Note, a host transformed with the expression vector,was designated as Escherichia coli SBM 308, and deposited with theFermentation Research Institute, Agency of Industrial Science andTechnology, 1-3, Higashi 3-chome, Tsukuba-shi, Ibaraki, Japan as FERMP-11506 on Jun. 7, 1990, and transferred to an international depositionunder the Budapest treaty as FERM BP-4197 on Feb. 18, 1993.

The clone pdKCR-DHFR-TP055 containing megakaryocyte differentiationfactor cDNA which was incorporated into pdKCR-DHFR was sequenced using aTaq Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystem) and afluorescent sequencer (Applied Biosystem Type 370A) according toincluded instructions. As a result, the determined nucleotide sequenceconformed to the sequence of the nucleotide numbers 99 to 1236 of SEQ IDNO: 30 and oligomers NI078 and NI079. In addition, it was confirmed bythe sequencing that a megakaryocyte differentiation factor CDNA insertedinto the vector is in correct orientation in relation to an expressionvector promoter.

As shown in the above, once the information of SEQ ID NO: 30 isprovided, it is easy for a person skilled in the art that the nucleotidesequence is determined by amplifying cDNA coding for megakaryocytedifferentiation factor in total or in a optional portion onmegakaryocyte differentiation factor expressing cell line (for example,A431) and cloned in a optional expression vector.

Example 4

Expression of megakaryocyte differentiation factor in Bombyx mori

(1) Construction of Bombyx mori expression vector

A megakaryocyte differentiation factor cDNA clone pdKCR-DHFR-TP055 wasdigested with NotI to cleave the NotI recognizing site artificiallyadded to the NI079. The NotI cohesive end thus generated was blunt-endedusing a blunting kit available from Takara Shuzo, and to the blunt endwas added an XbaI linker (Takara Shuzo) according to an attachedinstruction. The plasmid thus obtained was digested simultaneously withNruI and XbaI to cleave the NruI recognizing site artificially added tothe NI078 and the XbaI recognizing site of the XbaI linker introduced toprepare a megakaryocyte differentiation factor cDNA fragment having anNurI cohesive end and an XbaI cohesive end. This DNA fragment wasinserted at the Nru I recognizing site into a baculovirus transfervector for Bombyx mori nuclear polyhedrosis virus, pBm4 (available fromDepartment of Insect Genetics and Bleeding National Institute ofSericultural and Entomological Science, Ohwashi, Tukuba, Ibaraki 305,Japan) simultaneously digested with NruI and XbaI to obtain pBm4-TP055.

(2) Construction of TP055 recombinant virus

A cell line derived from Bombyx mori embryonic, BoMo15AIIc (availablefrom Department of Insect Genetics and Bleeding National Institute ofSericultural and Entomological Science, Ohwashi, Tukuba, Ibaraki 305,Japan) was subcultured in a medium containing 10% fetal bovine serum(FBS: GIBCO BRL) and 500 μg/ml gentamicin in MGM 448 at 25° C. TP055recombinant virus was constructed by co-introducing Bombyx mori nuclearpolyhedrosis virus gene DNA and pBm4-TP055 plasmid DNA into Bombyx moricultured cells by, for example, calcium phosphate co-precipitationmethod.

Namely, 2 μg of genomic DNA of wild type virus B6E (available fromDepartment of Insect Genetics and Bleeding National Institute ofSericultural and Entomological Science, Ohwashi, Tukuba, Ibaraki 305,Japan) and 10 μg of the transfer plasmid pBm4-TP055 were dissolved in240 μl of sterile purified water, and to the solution was added the samevolume of 0.5 M CaCl₂ and 0.1M HEPES, and the mixture was mixed andallowed to stand at a room temperature for 10 minutes. To the mixturewas added 480 μl of 0.2M NaCl, 0.05M HEPES, 0.75 mM NaH₂ PO₄ and 0.75 mMNa₂ HPO₄, and the mixture was stirred for a few second and allowed tostand at a room temperature for 20 to 30 minutes to form calciumphosphate gel containing the genomic DNA and the plasmid.

Next, 960 μl of calcium phosphate gel suspension containing the viralgenomic DNA and the transfer vector was added to 4 ml of BoMo15AIIccells in a 25 cm² T flask (T25, Corning), and the mixture was allowed tostand for 12 hours. The medium was replaced with a fresh MGM448(containing 10% FBS and the antibiotics), and culturing was carried outfor 25° C. On the sixth day the cultured medium was recovered as a viralsolution.

The cultured medium was centrifuged to obtain a clear supernatant, whichwas then diluted, and added to BoMo15AIIc cells cultured on themicrotiter plate, and after 8 days a culturing medium in which viralinfection was microscopically observed but a polyhedral body was notformed, was selected (by a limited dilution method). The cultured mediumwas recovered. Contamination with a wild virus in the viral solutionfactor was not observed.

A recombinant virus thus constructed, containing a DNA coding formegakaryocyte differentiation factor was designated as TP055-BmNPV.

(3) Test for expression of recombinant gene About 1×10⁶ BoMo15A IIccells were cultured in 4 ml of MGM448 medium containing 10% FBS on thebottom of 25 Cm² area of a flask for 2 days by plate culture. To theculture, 0.5 moi of wild type virus B6E or recombinant virus(TP055-BmNPV) containing a gene coding for megakaryocyte differentiationfactor were added and, BoMo15A IIc cells infected, and the cells werecultured at 25° C. for 3 days, and total RNA was extracted using Isogen(Wako Pure Chemical). Similarly, total RNA was extracted fromnon-infected BoMo 15AIIc cells.

Next, 1 mg of the RNA thus extracted was size-fractionated by agarosegel electrophoresis, and the separated RNA was transferred to aZetaprobe membrane by the capillary action. The membrane was soaked in ahybridization buffer containing megakaryocyte differentiation factorcDNA (:PCR product amplified with KY100 and NIO65 described in Example2.1) (TPO55 probe DNA) labeled with digoxigenin (Boehringer Mannheim),and the mixture was incubated at 42° C. for 12 hours to allow theformation of specific complex of recombinant megakaryocytedifferentiation factor mRNA and the TP055 probe DNA thereof. The complexwas then reacted with an alkaline phosphatase-conjugatedanti-digoxigenin antibody (Boehringer Mannheim), and the complexedmegakaryocyte differentiation factor mRNA was detected bychemoluminescence generated by hydrolysis of Lumigen PPD (AMPPD)(Boehringer Mannheim) according to manufacture's instructions withalkaline phosphates.

As seen in FIG. 11, recombinant megakaryocyte differentiation factormRNA was detected in total RNA extracted from TP055-BmNPV-infectedcells, and it was shown that the mRNA was expressed in theTP055-BmNPV-infected cells. On the other hand expression of mRNA whichhydridized with the probe DNA was not observed in the B6E-infected cellsand uninfected cells.

(4) Preparation of solution of recombinant virus

About 1×10⁶ BoMo15AIIc cells were cultured in 4 ml of MGM448 containing10% FBS on the bottom of a 25 cm² flask for 2 days, and to this culturewas added 10 μl of the culture medium of BoMo15AIIc cells containing therecombinant virus cloned in the above section (2). After culturing at25° C. for 14 days, the culture medium was centrifuged at 1000 rpm for 5minutes to obtain a supernatant as a recombinant virus solution.

(5) Preparation of hemolymph of Bombyx mori

50 μl/head of a viral solution of the 10⁻¹ -diluted recombinant virussolution obtained in the above section (3) or a 10⁻¹ -diluted wild typevirus B6E solution was injected to Bombyx mori larvae at 5th instar, andthe silkworms were fed with commercially available artificial feed(Morus; Katakura Kogyo) at 20° C. for 4 to 5 days. The abdomens of 50silkworms were cut, and an extract containing the hemolymph and thecontent in the central intestine was taken in a plastic tube cooled withice, and centrifuged to obtain a supernatant.

(6) Confirmation of activity of megakaryocyte differentiation factor

50 ml of the hemolymph of the silkworms obtained in the above section(5) was thoroughly dialyzed against a 20 mM Tris/HCl (pH 7.4) buffer,and applied to a Matrex Blue A column (φ2.5×15 cm) equilibrated with thesame buffer. The column was thoroughly washed with the same buffer toeliminate an unbound fraction, and a bound protein was eluted by aconcentration gradient of 0 to 1M NaCl. An elution profile formegakaryocyte differentiation activity of the hemolymph obtained fromsilkworms injected with the recombinant virus was compared with that fora wild type virus.

As seen from FIG. 12, megakaryocyte differentiation activity in thehemolymph from silkworms injected with the recombinant virus wassignificantly higher than that for the wild type virus.

Although Bombyx mori baculovirus transfer vector pBm4, Bombyx morinuclear polyhedrosis virus PbE and Bombyx mori cells BoMo15AIIc wereused in Example 4, the present invention is not limited to the use ofthese materials. Namely, other baculovirus transfer vector (such aspBK283, pBKblue, available from Funakoshi) Bombyx mori nuclearpolyhedrosis virus (such as purified DNA available from Funakoshi),Bombyx mori cells (such as BmN4 cells, available from Funakoshi) can beeasily used by a person with ordinary skill in the art to obtain amegakaryocyte differentiation factor.

A megakaryocyte differentiation factor of the present inventionaccelerates formation of megakaryocytes from myeloid cells in thepresence of IL-3. The present megakaryocyte differentiation factor playsan important role in differentiation of megakaryocytes and acts in vivoas a thrombopoietin. Accordingly, the present megakaryocytedifferentiation factor may be medicaments effective to not only variousdiseases involving decrease of platelets but also for control of thenumber of platelets decreasing by radiation in the case of bone marrowtransradiation, or for control of the number of platelets inchemotherapy of cancers.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 34                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ser Glu Thr Ile Asn Cys His Phe - # Lys                                #  5 1                                                                        - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Trp Gln Ser Ala Phe Thr Lys                                            #  5 1                                                                        - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Val Glu Arg Val Asp Phe Thr Asn - # His Leu Glu Asp Thr Arg Arg        Asn                                                                           #   15                                                                        -      Ile Asn Lys                                                            - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ser Tyr Ile Glu Val Thr Glu Glu - # Gly Thr Glu Ala Thr Ala Ala        Thr                                                                           #   15                                                                        -      Gly                                                                    - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Gln Tyr Leu Arg Ala Leu Gly Leu - # Lys                                #  5 1                                                                        - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Asp Leu Ser Gly Ile Ala Ser - # Gly Gly Arg Leu Tyr Ile Ser        Arg                                                                           #   15                                                                        -      Met Met Gly Lys                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Leu Tyr Asp Ala Lys                                                    #  5 1                                                                        - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Asn Tyr Glu Met Lys                                                    #  5 1                                                                        - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Val Ala Met Met His Gln Glu - # Arg Lys                            #   10                                                                        - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 3..31                                                 #/note= "Corresponding to amino acid                                          #of SEQ ID NO: 3; N is inosine."                                              #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ                                      #     38           TYAC NAAYCAYYTN GARGAYAC                                   - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 9..28                                                 #/note= "Corresponding to amino acid                                          #of SEQ ID NO: 4; N is inosine."                                              #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ                                      #          32      ARGA RGGNACNGAR GC                                         - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..37                                                 #/note= "Oligomer attached toON:                                              #kit (Gibco BRL)."RACE                                                        #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ                                      #      37          GTAC TTTTTTTTTT TTTTTTT                                    - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               TATA                                                       - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                23TAAG TAT                                                   - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                GAGA G                                                     - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                TACA T                                                     - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                GCCC G                                                     - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                 22TAA TG                                                    - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       36         AGCC GGCCGCCAGA GGCAAT                                     - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                ACCA G                                                     - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:21:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24GCCT GAAG                                                  - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ                                      #             51TACAGCC GGCCGCCAGA GGCAATGCCA GACAGGTCAG C                    - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ                                      #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ                                      #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:25:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               AGTG                                                       - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:26:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               AACA                                                       - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:27:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                AAGT A                                                     - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                ACTG C                                                     - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:29:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                ATTA C                                                     - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1950 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                                (B) STRAIN: A431                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1                                                     #/note= "DNA coding for humanON:                                                             megakaryocyt - #e differentiation factor."                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 74..1217                                              #ID NO:30:(xi) SEQUENCE DESCRIPTION: SEQ                                      - GGCACGAGAG GAACTGAAGC CCAGCTGTGA AGGCCGCAGA CTGCAGTGAG AG - #GAGGCTGC         60                                                                          - ACTCCATTTT GCA ATG GCC TCC CTT GCT GCA GCA AA - #T GCA GAG TTT TGC           109                                                                          #Ser Leu Ala Ala Ala Asn Ala Glu Phe Cys                                      #              10                                                             - TTC AAC CTG TTC AGA GAG ATG GAT GAC AAT CA - #A GGA AAT GGA AAT GTG          157                                                                          Phe Asn Leu Phe Arg Glu Met Asp Asp Asn Gl - #n Gly Asn Gly Asn Val           #         25                                                                  - TTC TTT TCC TCT CTG AGC CTC TTC GCT GCC CT - #G GCC CTG GTC CGC TTG          205                                                                          Phe Phe Ser Ser Leu Ser Leu Phe Ala Ala Le - #u Ala Leu Val Arg Leu           #     40                                                                      - GGC GCT CAA GAT GAC TCC CTC TCT CAG ATT GA - #T AAG TTG CTT CAT GTT          253                                                                          Gly Ala Gln Asp Asp Ser Leu Ser Gln Ile As - #p Lys Leu Leu His Val           # 60                                                                          - AAC ACT GCC TCA GGA TAT GGA AAC TCT TCT AA - #T AGT CAG TCA GGG CTC          301                                                                          Asn Thr Ala Ser Gly Tyr Gly Asn Ser Ser As - #n Ser Gln Ser Gly Leu           #                 75                                                          - CAG TCT CAA CTG AAA AGA GTT TTT TCT GAT AT - #A AAT GCA TCC CAC AAG          349                                                                          Gln Ser Gln Leu Lys Arg Val Phe Ser Asp Il - #e Asn Ala Ser His Lys           #             90                                                              - GAT TAT GAT CTC AGC ATT GTG AAT GGG CTT TT - #T GCT GAA AAA GTG TAT          397                                                                          Asp Tyr Asp Leu Ser Ile Val Asn Gly Leu Ph - #e Ala Glu Lys Val Tyr           #        105                                                                  - GGC TTT CAT AAG GAC TAC ATT GAG TGT GCC GA - #A AAA TTA TAC GAT GCC          445                                                                          Gly Phe His Lys Asp Tyr Ile Glu Cys Ala Gl - #u Lys Leu Tyr Asp Ala           #   120                                                                       - AAA GTG GAG CGA GTT GAC TTT ACG AAT CAT TT - #A GAA GAC ACT AGA CGT          493                                                                          Lys Val Glu Arg Val Asp Phe Thr Asn His Le - #u Glu Asp Thr Arg Arg           125                 1 - #30                 1 - #35                 1 -       #40                                                                           - AAT ATT AAT AAG TGG GTT GAA AAT GAA ACA CA - #T GGC AAA ATC AAG AAC          541                                                                          Asn Ile Asn Lys Trp Val Glu Asn Glu Thr Hi - #s Gly Lys Ile Lys Asn           #               155                                                           - GTG ATT GGT GAA GGT GGC ATA AGC TCA TCT GC - #T GTA ATG GTG CTG GTG          589                                                                          Val Ile Gly Glu Gly Gly Ile Ser Ser Ser Al - #a Val Met Val Leu Val           #           170                                                               - AAT GCT GTG TAC TTC AAA GGC AAG TGG CAA TC - #A GCC TTC ACC AAG AGC          637                                                                          Asn Ala Val Tyr Phe Lys Gly Lys Trp Gln Se - #r Ala Phe Thr Lys Ser           #       185                                                                   - GAA ACC ATA AAT TGC CAT TTC AAA TCT CCC AA - #G TGC TCT GGG AAG GCA          685                                                                          Glu Thr Ile Asn Cys His Phe Lys Ser Pro Ly - #s Cys Ser Gly Lys Ala           #   200                                                                       - GTC GCC ATG ATG CAT CAG GAA CGG AAG TTC AA - #T TTG TCT GTT ATT GAG          733                                                                          Val Ala Met Met His Gln Glu Arg Lys Phe As - #n Leu Ser Val Ile Glu           205                 2 - #10                 2 - #15                 2 -       #20                                                                           - GAC CCA TCA ATG AAG ATT CTT GAG CTC AGA TA - #C AAT GGT GGC ATA AAC          781                                                                          Asp Pro Ser Met Lys Ile Leu Glu Leu Arg Ty - #r Asn Gly Gly Ile Asn           #               235                                                           - ATG TAC GTT CTG CTG CCT GAG AAT GAC CTC TC - #T GAA ATT GAA AAC AAA          829                                                                          Met Tyr Val Leu Leu Pro Glu Asn Asp Leu Se - #r Glu Ile Glu Asn Lys           #           250                                                               - CTG ACC TTT CAG AAT CTA ATG GAA TGG ACC AA - #T CCA AGG CGA ATG ACC          877                                                                          Leu Thr Phe Gln Asn Leu Met Glu Trp Thr As - #n Pro Arg Arg Met Thr           #       265                                                                   - TCT AAG TAT GTT GAG GTA TTT TTT CCT CAG TT - #C AAG ATA GAG AAG AAT          925                                                                          Ser Lys Tyr Val Glu Val Phe Phe Pro Gln Ph - #e Lys Ile Glu Lys Asn           #   280                                                                       - TAT GAA ATG AAA CAA TAT TTG AGA GCC CTA GG - #G CTG AAA GAT ATC TTT          973                                                                          Tyr Glu Met Lys Gln Tyr Leu Arg Ala Leu Gl - #y Leu Lys Asp Ile Phe           285                 2 - #90                 2 - #95                 3 -       #00                                                                           - GAT GAA TCC AAA GCA GAT CTC TCT GGG ATT GC - #T TCG GGG GGT CGT CTG         1021                                                                          Asp Glu Ser Lys Ala Asp Leu Ser Gly Ile Al - #a Ser Gly Gly Arg Leu           #               315                                                           - TAT ATA TCA AGG ATG ATG CAC AAA TCT TAC AT - #A GAG GTC ACT GAG GAG         1069                                                                          Tyr Ile Ser Arg Met Met His Lys Ser Tyr Il - #e Glu Val Thr Glu Glu           #           330                                                               - GGC ACC GAG GCT ACT GCT GCC ACA GGA AGT AA - #T ATT GTA GAA AAG CAA         1117                                                                          Gly Thr Glu Ala Thr Ala Ala Thr Gly Ser As - #n Ile Val Glu Lys Gln           #       345                                                                   - CTC CCT CAG TCC ACG CTG TTT AGA GCT GAC CA - #C CCA TTC CTA TTT GTT         1165                                                                          Leu Pro Gln Ser Thr Leu Phe Arg Ala Asp Hi - #s Pro Phe Leu Phe Val           #   360                                                                       - ATC AGG AAG GAT GAC ATC ATC TTA TTC AGT GG - #C AAA GTT TCT TGC CCT         1213                                                                          Ile Arg Lys Asp Asp Ile Ile Leu Phe Ser Gl - #y Lys Val Ser Cys Pro           365                 3 - #70                 3 - #75                 3 -       #80                                                                           - TGA A AATCCAATTG GTTTCTGTTA TAGCAGTCCC CACAACATCA AAGA - #ACCACC            1267                                                                           *                                                                            - ACAAGTCAAT AGATTTGAGT TTAATTGGAA AAATGTGGTG TTTCCTTTGA GT - #TTATTTCT       1327                                                                          - TCCTAACATT GGTCAGCAGA TGACACTGGT GACTTGACCC TTCCTAGACA CC - #TGGTTGAT       1387                                                                          - TGTCCTGATC CCTGCTCTTA GCATTCTACC ACCATGTGTC TCACCCATTT CT - #AATTTCAT       1447                                                                          - TGTCTTTCTT CCCACGCTCA TTTCTATCAT TCTCCCCCAT GACCCGTCTG GA - #AATTATGG       1507                                                                          - AGAGTGCTCA ACTGGTAAGG AGAACGTAGA AGTAGCCCTA GGGATCCTTT TT - #GAAACTCT       1567                                                                          - ACAGTTATCG CAGATATTCT AGCTTCATTG TAAGCAATCT AGGAAATAAG CC - #CTGCTGCT       1627                                                                          - TTCTAGAAAT AAGTGTGAAG GATAAATTTT CTTTGTTGAC CTATGAAGAT TT - #TAGAGTTT       1687                                                                          - ACCTTCATAT GTTTGATTTT AAATCAGTGT ATAATCTAGA TGGTAAAAAA TG - #TGAAATTG       1747                                                                          - GGATTAGGGA CCAACCAAAA TATTTCATTA ATGCTTTCAA TTGACAAATT TT - #GGTCTTTC       1807                                                                          - TTTGATAAGA CAATATGTAC ATAGTTTTTT CAAATATTAA AGATCTTTTA AC - #TGTTGGCA       1867                                                                          - GTTGTTATCT ACAGAATCAT ATCTCATATG CTGTGTAGTT TATAAGTTTT TT - #CTCTATTT       1927                                                                          #              1950ACAA CAT                                                   - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1                                                     #/note= "5'-non-translation region."                                          #ID NO:31:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               TGAA                                                       - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:32:(xi) SEQUENCE DESCRIPTION: SEQ                                      #      37          CCTC CCTTGCTGCA GCAAATG                                    - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 49 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:33:(xi) SEQUENCE DESCRIPTION: SEQ                                      #               49CGTGG TGGTTCTTTG ATGTTGTGGG GACTGCTAT                       - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:  380 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:34:                      - Met Ala Ser Leu Ala Ala Ala Asn Ala Glu Ph - #e Cys Phe Asn Leu Phe         #                 15                                                          - Arg Glu Met Asp Asp Asn Gln Gly Asn Gly As - #n Val Phe Phe Ser Ser         #             30                                                              - Leu Ser Leu Phe Ala Ala Leu Ala Leu Val Ar - #g Leu Gly Ala Gln Asp         #         45                                                                  - Asp Ser Leu Ser Gln Ile Asp Lys Leu Leu Hi - #s Val Asn Thr Ala Ser         #     60                                                                      - Gly Tyr Gly Asn Ser Ser Asn Ser Gln Ser Gl - #y Leu Gln Ser Gln Leu         # 80                                                                          - Lys Arg Val Phe Ser Asp Ile Asn Ala Ser Hi - #s Lys Asp Tyr Asp Leu         #                 95                                                          - Ser Ile Val Asn Gly Leu Phe Ala Glu Lys Va - #l Tyr Gly Phe His Lys         #           110                                                               - Asp Tyr Ile Glu Cys Ala Glu Lys Leu Tyr As - #p Ala Lys Val Glu Arg         #       125                                                                   - Val Asp Phe Thr Asn His Leu Glu Asp Thr Ar - #g Arg Asn Ile Asn Lys         #   140                                                                       - Trp Val Glu Asn Glu Thr His Gly Lys Ile Ly - #s Asn Val Ile Gly Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Gly Ile Ser Ser Ser Ala Val Met Val Le - #u Val Asn Ala Val Tyr         #               175                                                           - Phe Lys Gly Lys Trp Gln Ser Ala Phe Thr Ly - #s Ser Glu Thr Ile Asn         #           190                                                               - Cys His Phe Lys Ser Pro Lys Cys Ser Gly Ly - #s Ala Val Ala Met Met         #       205                                                                   - His Gln Glu Arg Lys Phe Asn Leu Ser Val Il - #e Glu Asp Pro Ser Met         #   220                                                                       - Lys Ile Leu Glu Leu Arg Tyr Asn Gly Gly Il - #e Asn Met Tyr Val Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Pro Glu Asn Asp Leu Ser Glu Ile Glu As - #n Lys Leu Thr Phe Gln         #               255                                                           - Asn Leu Met Glu Trp Thr Asn Pro Arg Arg Me - #t Thr Ser Lys Tyr Val         #           270                                                               - Glu Val Phe Phe Pro Gln Phe Lys Ile Glu Ly - #s Asn Tyr Glu Met Lys         #       285                                                                   - Gln Tyr Leu Arg Ala Leu Gly Leu Lys Asp Il - #e Phe Asp Glu Ser Lys         #   300                                                                       - Ala Asp Leu Ser Gly Ile Ala Ser Gly Gly Ar - #g Leu Tyr Ile Ser Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Met Met His Lys Ser Tyr Ile Glu Val Thr Gl - #u Glu Gly Thr Glu Ala         #               335                                                           - Thr Ala Ala Thr Gly Ser Asn Ile Val Glu Ly - #s Gln Leu Pro Gln Ser         #           350                                                               - Thr Leu Phe Arg Ala Asp His Pro Phe Leu Ph - #e Val Ile Arg Lys Asp         #       365                                                                   - Asp Ile Ile Leu Phe Ser Gly Lys Val Ser Cy - #s Pro                         #   380                                                                       __________________________________________________________________________

We claim:
 1. An isolated, purified megakaryocyte differentiation factorhaving the following properties;(1) stimulating differentiation ofmegakaryocytes; (2) exhibiting a molecular weight of 55 to 57 kD asdetermined by gel filtration and SDS-polyacrylamide gel electrophoresis(SDS-PAGE), and having no intermolecular disulfide linkage; (3)exhibiting an isoelectric point of 6.5±0.5; and (4) having at least oneof the amino acid sequences shown in SEQ ID NO: 1 to 9 in the SequenceListing.
 2. An isolated, purified megakaryocyte differentiation factoraccording to claim 1, produced by human cells.
 3. An isolated, purifiedmegakaryocyte differentiation factor according to claim 2, produced byhuman cancer cells.
 4. An isolated, purified megakaryocytedifferentiation factor according to claim 3, produced by cells capableof growing in a protein-free medium, wherein said cells are derived fromhuman epidermoid carcinoma cell A431.
 5. An isolated, purifiedmegakaryocyte differentiation factor according to claim 4, produced bycells capable of growing in a protein-free medium, wherein said cellsare derived from human epidermoid carcinoma cell A431, cultured in aprotein-free medium.
 6. A pharmaceutical composition comprising amegakaryocyte differentiation factor according to claim 1 and apharmaceutically acceptable carrier therefor.
 7. A pharmaceuticalcomposition according to claim 6, comprising an amount of megakaryocytedifferentiation factor effective for stimulating differentiation ofmegakaryocytes, for treating thrombocytopenia.
 8. An isolated, purifiedmegakaryocyte differentiation factor according to claim 1, includingeach of the amino acid sequences shown in SEQ ID NOs: 1 to
 9. 9. Arecombinant megakaryocyte differentiation factor consisting of the aminoacid sequence shown in SEQ ID NO:
 34. 10. A megakaryocytedifferentiation factor according to claim 9, which is glycosylated. 11.A megakaryocyte differentiation factor according to claim 9, wherein theN-terminus of the factor is biochemically or chemically modified,wherein the first amino acid methionine is deleted, the second aminoacid alanine is acetylated, or both.
 12. A pharmaceutical compositioncomprising a megakaryocyte differentiation factor according to claim 11and a pharmaceutically acceptable carrier therefor.
 13. A recombinantmegakaryocyte differentiation factor consisting of an amino acidsequence selected from the group consisting of: the amino acid sequenceshown in SEQ ID NO: 34; an amino acid sequence wherein one to thirtyamino acid residues in the amino acid sequence shown in SEQ ID NO: 34are deleted; an amino acid sequence wherein one to ten amino acidresidues in the amino acid sequence shown in SEQ ID NO: 34 are replacedwith at least one other amino acid; an amino acid sequence wherein atleast one amino acid is added to the amino acid sequence shown in SEQ IDNO: 34; and an amino acid sequence including a combination of said aminoacid modifications.
 14. A megakaryocyte differentiation factor accordingto claim 13, which is glycosylated.
 15. A megakaryocyte differentiationfactor according to claim 13, wherein the N-terminus of the factor isbiochemically or chemically modified, such that the first amino acid,methionine, is deleted, the second amino acid, alanine, is acetylated,or both.
 16. A pharmaceutical composition comprising a megakaryocytedifferentiation factor according to claim 13 and a pharmaceuticallyacceptable carrier therefor.
 17. A pharmaceutical composition accordingto claim 16, which contains an amount of the megakaryocytedifferentiation factor effective for stimulating differentiation ofmegakaryocytes, for treating thrombocytopenia.
 18. An isolated, purifiedmegakaryocyte differentiation factor consisting of the amino acidsequence shown in SEQ ID NO:
 34. 19. A pharmaceutical compositioncomprising a megakaryocyte differentiation factor according to claim 18,and a pharmaceutically acceptable carrier therefor.